This invention relates to the use of N-formyl hydroxylamine derivatives as antibacterial agents, to a novel class of such compounds, and to pharmaceutical and veterinary compositions comprising such compounds.
In general, bacterial pathogens are classified as either Gram-positive or Gram-negative. Many antibacterial agents (including antibiotics) are specific against one or other Gram-class of pathogens. Antibacterial agents effective against both Gram-positive and Gram-negative pathogens are therefore generally regarded as having broad spectrum activity.
Many classes of antibacterial agents are known, including the penicillins and cephalosporins, tetracyclines, sulfonamides, monobactams, fluoroquinolones and quinolones, aminoglycosides, glycopeptides, macrolides, polymyxins, lincosamides, trimethoprim and chloramphenicol. The fundamental mechanisms of action of these antibacterial classes vary.
Bacterial resistance to many known antibacterials is a growing problem. Accordingly there is a continuing need in the art for alternative antibacterial agents, especially those which have mechanisms of action fundamentally different from the known classes.
Amongst the Gram-positive pathogens, such as Staphylococci, Streptococci, Mycobacteria and Enterococci, resistant strains have evolved/arisen which makes them particularly difficult to eradicate. Examples of such strains are methicillin resistant Staphylococcus aureus (MRSA), methicillin resistant coagulase negative Staphylococci (MRCNS), penicillin resistant Streptococcus pneumoniae and multiply resistant Enterococcus faecium. 
Pathogenic bacteria are often resistant to the aminoglycoside, xcex2-lactam (penicillins and cephalosporins), and chloramphenicol types of antibiotic. This resistance involves the enzymatic inactivation of the antibiotic by hydrolysis or by formation of inactive derivatives. The xcex2-lactam (penicillin and cephalosporin) family of antibiotics are characterised by the presence of a xcex2-lactam ring structure. Resistance to this family of antibiotics in clinical isolates is most commonly due to the production of a xe2x80x9cpenicillinasexe2x80x9d (xcex2-lactamase) enzyme by the resistant bacterium which hydrolyses the xcex2-lactam ring thus eliminating its antibacterial activity.
Recently there has been an emergence of vancomycin-resistant strains of enterococci (Woodford N. 1998 Glycopeptide-resistant enterococci: a decade of experience. Journal of Medical Microbiology. 47(10):849-62). Vancomycin-resistant enterococci are particularly hazardous in that they are frequent causes of hospital based infections and are inherently resistant to most antibiotics. Vancomycin works by binding to the terminal D-Ala-D-Ala residues of the cell wall peptidioglycan precursor. The high-level resistance to vancomycin is known as VanA and is conferred by a genes located on a transposable element which alter the terminal residues to D-Ala-D-lac thus reducing the affinity for vancomycin.
In view of the rapid emergence of multidrug-resistant bacteria, the development of antibacterial agents with novel modes of action that are effective against the growing number of resistant bacteria, particularly the vancomycin resistant enterococci and xcex2-lactam antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus, is of utmost importance.
This invention is based on the finding that certain N-formyl hydroxylamine derivatives have antibacterial activity, and makes available a new class of antibacterial agents. The inventors have found that the compounds with which this invention is concerned are antibacterial with respect to a range of Gram-positive and Gram-negative organisms. Furthermore, there is evidence that some compounds are antibacterial with respect to bacteria which are resistant to commonly used antibiotics such as vancomycin and the xcex2-lactam antibiotics, for example methicillin-resistant Staphylococcus aureus. 
Although it may be of interest to establish the mechanism of action of the compounds with which the invention is concerned, it is their ability to inhibit bacterial growth which makes them useful. However, it is presently believed that their antibacterial activity is due, at least in part, to intracellular inhibition of bacterial polypeptide deformylase (PDF) enzyme.
Bacterial polypeptide deformylases (PDF) (EC3.5.1.31), are a conserved family of metalloenzymes (Reviewed: Meinnel T, Lazennec C, Villoing S, Blanquet S, 1997, Journal of Molecular Biology 267, 749-761) which are essential for bacterial viability, their function being to remove the formyl group from the N-terminal methionine residue of ribosome-synthesised proteins in eubacteria. Mazel et al. (EMBO J. 13(4):91 4-923, 1994) have recently cloned and characterised an E. coli PDF. As PDF is essential to the growth of bacteria and there is no eukaryotic counterpart to PDF, Mazel et al. (ibid), Rajagopalan et al. (J. Am. Chem. Soc. 119:12418-12419, 1997) and Becker et al., (J. Biol Chem. 273(19):11413-11416, 1998) have each proposed that PDF is an excellent anti-bacterial target.
Certain N-formyl hydroxylamine derivatives have previously been claimed in the patent publications listed below, although very few examples of such compounds have been specifically made and described:
The pharmaceutical utility ascribed to the N-formyl hydroxylamine derivatives in those publications is the ability to inhibit matrix metalloproteinases (MMPs) and in some cases release of tumour necrosis factor (TNF), and hence the treatment of diseases or conditions mediated by those enzymes, such as cancer and rheumatoid arthritis. That prior art does not disclose or imply that N-formyl hydroxylamine derivatives have antibacterial activity.
In addition to these, U.S. Pat. No. 4,738,803 (Roques et al.) also discloses N-formyl hydroxylamine derivatives, however, these compounds are disclosed as enkephalinase inhibitors and are proposed for use as antidepressants and hypotensive agents. Also, WO 97/38705 (Bristol-Myers Squibb) discloses certain N-formyl hydroxylamine derivatives as enkephalinase and angiotensin converting enzyme inhibitors. This prior art does not disclose or imply that N-formyl hydroxylamine derivatives have antibacterial activity either.
According to the first aspect of the present invention there is provided the use of a compound of formula (I) or a pharmaceutically or veterinarily acceptable salt thereof in the preparation of an antibacterial composition: 
wherein:
R1 represents hydrogen, or C1-C6 alkyl or C1-C6 alkyl substituted by one or more halogen atoms;
R2 represents a group R10xe2x80x94(X)nxe2x80x94(ALK)mxe2x80x94 wherein
R10 represents hydrogen, or a C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, aryl, or heterocyclyl group, any of which may be unsubstituted or substituted by (C1-C6)alkyl, (C1-C6)alkoxy, hydroxy, mercapto, (C1-C6)alkylthio, amino, halo (including fluoro, chloro, bromo and iodo), trifluoromethyl, cyano, nitro, xe2x80x94COOH, xe2x80x94CONH2, xe2x80x94COORA, xe2x80x94NHCORA, xe2x80x94CONHRA, xe2x80x94NHRA, xe2x80x94NRARB, or xe2x80x94CONRARB wherein RA and RB are independently a (C1-C6)alkyl group, and
ALK represents a straight or branched divalent C1-C6 alkylene, C2-C6 alkenylene, or C2-C6 alkynylene radical, and may be interrupted by one or more non-adjacent xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94 linkages,
X represents xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, and
m and n are independently 0 or 1; and
A represents (i) a group of formula (IA), (IB), (IC) or (ID) 
wherein:
R3 represents hydrogen and R4 represents the side chain of a natural or non-natural alpha amino acid or R3 and R4 when taken together with the nitrogen and carbon atoms to which they are respectively attached form an optionally substituted saturated heterocyclic ring of 5 to 8 atoms which ring is optionally fused to a carbocyclic or second heterocyclic ring,
R5 and R6, independently represent hydrogen, or optionally substituted C1-C8 alkyl, cycloalkyl, aryl, aryl(C1-C6 alkyl), heterocyclic, or heterocyclic(C1-C6 alkyl), or R5 and R6 when taken together with the nitrogen atom to which they are attached form an optionally substituted saturated heterocyclic ring of 3 to 8 atoms which ring is optionally fused to a carbocyclic or second heterocyclic ring, and
R7 represents hydrogen, C1-C6 alkyl, or an acyl group.
In another aspect, the invention provides a method for the treatment of bacterial infections in humans and non-human mammals, which comprises administering to a subject suffering such infection an antibacterially effective dose of a compound of formula (I) as defined above.
In a further aspect of the invention there is provided a method for the treatment of bacterial contamination by applying an antibacterially effective amount of a compound of formula (I) as defined above to the site of contamination.
The compounds of formula (I) as defined above may be used as component(s) of antibacterial cleaning or disinfecting materials.
According to a preferred embodiment, the various aspects of the invention can be applied against vancomycin-, quinolone- and xe2x80x9cxcex2-lactamxe2x80x9d-resistant bacteria and the infections they cause.
On the hypothesis that the compounds (I) act by inhibition of intracellular PDF, the most potent antibacterial effect may be achieved by using compounds which efficiently pass through the bacterial cell wall. Thus, compounds which are highly active as inhibitors of PDF in vitro and which penetrate bacterial cells are preferred for use in accordance with the invention. It is to be expected that the antibacterial potency of compounds which are potent inhibitors of the PDF enzyme in vitro, but are poorly cell penetrant, may be improved by their use in the form of a prodrug, ie a structurally modified analogue which is converted to the parent molecule of formula (I), for example by enzymic action, after it has passed through the bacterial cell wall.
The invention also provides novel compounds of formula (I) above, or pharmaceutically or veterinarily acceptable salts thereof, wherein:
R1 represents hydrogen, C1-C6 alkyl or C1-C6 alkyl substituted by one or more halogen atoms;
R2 represents a group R10xe2x80x94(ALK)mxe2x80x94 wherein
R10 represents hydrogen, or a C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, a cycloalkyl, aryl, or heterocyclyl group, any of which may be unsubstituted or substituted by (C1-C6)alkyl, (C1-C6)alkoxy, hydroxy, mercapto, (C1-C6)alkylthio, amino, halo (including fluoro, chloro, bromo and iodo), trifluoromethyl, nitro, xe2x80x94COOH, xe2x80x94CONH2, xe2x80x94COORA, xe2x80x94NHCORA, xe2x80x94CONHRA, xe2x80x94NHRA, xe2x80x94NRARB, or xe2x80x94CONRARB wherein RA and RB are independently a (C1-C6)alkyl group,
ALK represents a straight or branched divalent C1-C6 alkylene, C2-C6 alkenylene, C2-C6 alkynylene radical, and may be interrupted by one or more non-adjacent xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94 linkages, and
m represents 0 or 1;
A represents a group of formula (IA), (IB), (IC) or (ID) above wherein:
R3 represents hydrogen and R4 represents the side chain of a natural or non-natural alpha amino acid or R3 and R4 when taken together with the nitrogen and carbon atoms to which they are respectively attached form an optionally substituted saturated heterocyclic ring of 5 to 8 atoms which ring is optionally fused to a carbocyclic or second heterocyclic ring,
R5 and R6, independently represent hydrogen, or optionally substituted C1-C8 alkyl, cycloalkyl, aryl(C1-C6 alkyl), non-aromatic heterocyclic, or heterocyclic(C1-C6 alkyl), or R5 and R6 when taken together with the nitrogen atom to which they are attached form an optionally substituted saturated heterocyclic ring of 3 to 8 atoms which ring is optionally fused to a carbocyclic or second heterocyclic ring, and
R7 represents hydrogen, C1-C6 alkyl, or an acyl group.
Provided that
(i) when A is a group of formula (IA) or (IB) and R2 is C2-C5 alkyl then R4 is not the side chain of a natural alpha amino acid or the side chain of a natural alpha-amino acid in which any functional substituents are protected, any amino groups are acylated, and any carboxyl groups are esterified;
(ii) when A is a group of formula (IA) or (IB) then R4 is not a bicyclicarylmethyl group; and
(iii) when A is a group of formula (IA) and R2 is cyclopropylmethyl, cyclobutylmethyl or cyclopentylmethyl and one of R5 and R6 is hydrogen, then R4 is not tert-butyl.
As used herein the term xe2x80x9c(C1-C6)alkylxe2x80x9d means a straight or branched chain alkyl moiety having from 1 to 6 carbon atoms, including for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n-hexyl.
As used herein the term xe2x80x9cdivalent (C1-C6)alkylene radicalxe2x80x9d means a saturated hydrocarbon chain having from 1 to 6 carbon atoms and two unsatisfied valencies.
As used herein the term xe2x80x9c(C2-C6)alkenylxe2x80x9d means a straight or branched chain alkenyl moiety having from 2 to 6 carbon atoms having at least one double bond of either E or Z stereochemistry where applicable. The term includes, for example, vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl.
As used herein the term xe2x80x9cdivalent (C2-C6)alkenylene radicalxe2x80x9d means a hydrocarbon chain having from 2 to 6 carbon atoms, at least one double bond, and two unsatisfied valencies.
As used herein the term xe2x80x9cC2-C6 alkynylxe2x80x9d refers to straight chain or branched chain hydrocarbon groups having from two to six carbon atoms and having in addition one triple bond. This term would include for example, ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.
As used herein the term xe2x80x9cdivalent (C2-C6)alkynylene radicalxe2x80x9d means a hydrocarbon chain having from 2 to 6 carbon atoms, at least one triple bond, and two unsatisfied valencies.
As used herein the term xe2x80x9ccycloalkylxe2x80x9d means a saturated alicyclic moiety having from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
As used herein the term xe2x80x9ccycloalkenylxe2x80x9d means an unsaturated alicyclic moiety having from 3-8 carbon atoms and includes, for example, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl. In the case of cycloalkenyl rings of from 5-8 carbon atoms, the ring may contain more than one double bond.
As used herein the term xe2x80x9carylxe2x80x9d refers to a mono-, bi- or tri-cyclic carbocyclic aromatic group, and to groups consisting of two covalently linked monocyclic carbocyclic aromatic groups. Illustrative of such groups are phenyl, biphenyl and napthyl.
As used herein the term xe2x80x9cheteroarylxe2x80x9d refers to a 5- or 6-membered aromatic ring containing one or more heteroatoms, and optionally fused to a benzyl or pyridyl ring; and to groups consisting of two covalently linked 5- or 6-membered aromatic rings each containing one or more heteroatoms; and to groups consisting of a monocyclic carbocyclic aromatic group covalently linked to a 5- or 6-membered aromatic rings containing one or more heteroatoms;. Illustrative of such groups are thienyl, furyl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, 4-([1,2,3]-thiadiazoly-4-yl)phenyl and 5-isoxazol-3-ylthienyl.
As used herein the unqualified term xe2x80x9cheterocyclylxe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d includes xe2x80x9cheteroarylxe2x80x9d as defined above, and in particular means a 5-7 membered aromatic or non-aromatic heterocyclic ring containing one or more heteroatoms selected from S, N and O, and optionally fused to a benzene ring, including for example, pyrrolyl, furyl, thienyl, piperidinyl, imidazolyl, oxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, benzimidazolyl, maleimido, succinimido, phthalimido and 1,3-dioxo-1,3-dihydro-isoindol-2-yl groups.
As used herein the term xe2x80x9cacylxe2x80x9d means a group R20C(O)xe2x80x94 where R20 is (C1-C6)alkyl, (C2-C6)alkenyl, (C3-C7)cycloalkyl, phenyl, heterocyclyl, phenyl(C1-C6)alkyl, heterocyclyl(C1-C6)alkyl, (C3-C7)cycloalkyl(C1-C6)alkyl, phenyl(C2-C6)alkenyl, heterocyclyl(C2-C6)alkenyl, (C3-C7)cycloalkyl(C2-C6)alkenyl, any of which R20 groups may be substituted.
As used herein, the term xe2x80x9cbicyclicarylmethylxe2x80x9d means (i) a methyl group substituted by a monocyclic aryl or heteroaryl group which in turn is substituted by a monocyclic aryl or heteroaryl group, or (ii) a methyl group substituted by a monocyclic aryl or heteroaryl group to which is fused a second monocyclic aryl or heteroaryl group; and includes both unsubstituted and substituted bicyclicarylmethyl. Examples of such bicyclicarylmethyl groups include naphthyl, indolyl, quinolyl and isoquinolyl.
Unless otherwise specified in the context in which it occurs, the term xe2x80x9csubstitutedxe2x80x9d as applied to any moiety herein means substituted with up to four substituents, each of which independently may be (C1-C6)alkyl, benzyl, (C1-C6)alkoxy, phenoxy, hydroxy, mercapto, (C1-C6)alkylthio, amino, halo (including fluoro, chloro, bromo and iodo), trifluoromethyl, nitro, xe2x80x94COOH, xe2x80x94CONH2, xe2x80x94CORA, xe2x80x94COORA, xe2x80x94NHCORA, xe2x80x94CONHRA, xe2x80x94NHRA, xe2x80x94NRARB, or xe2x80x94CONRARB wherein RA and RB are independently a (C1-C6)alky group. In the case where xe2x80x9csubstitutedxe2x80x9d means benzyl, the phenyl ring thereof may itself be substituted with any of the foregoing, except benzyl.
As used herein the terms xe2x80x9cside chain of a natural alpha-amino acidxe2x80x9d and xe2x80x9cside chain of a non-natural alpha-amino acidxe2x80x9d mean the group Rx in respectively a natural and non-natural amino acid of formula NH2xe2x80x94CH(Rx)xe2x80x94COOH.
Examples of side chains of natural alpha amino acids include those of alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, histidine, 5-hydroxylysine, 4-hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, xcex1-aminoadipic acid, xcex1-amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine, xcex1-methylserine, ornithine, pipecolic acid, and thyroxine.
In natural alpha-amino acid side chains which contain functional substituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups as in arginine, lysine, glutamic acid, aspartic acid, tryptophan, histidine, serine, threonine, tyrosine, and cysteine, such functional substituents may optionally be protected.
Likewise, in the side chains of non-natural alpha amino acids which contain functional substituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups, such functional substituents may optionally be protected.
The term xe2x80x9cprotectedxe2x80x9d when used in relation to a functional substituent in a side chain of a natural or non-natural alpha-amino acid means a derivative of such a substituent which is substantially non-functional. The widely used handbook by T. W. Greene and P. G. Wuts xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d Second Edition, Wiley, New York, 1991 reviews the subject. For example, carboxyl groups may be esterified (for example as a C1-C6 alkyl ester), amino groups may be converted to amides (for example as a NHCOC1-C6 alkyl amide) or carbamates (for example as an NHC(xe2x95x90O)OC1-C6 alkyl or NHC(xe2x95x90O)OCH2Ph carbamate), hydroxyl groups may be converted to ethers (for example an OC1-C6 alkyl or a O(C1-C6 alkyl)phenyl ether) or esters (for example a OC(xe2x95x90O)C1-C6 alkyl ester) and thiol groups may be converted to thioethers (for example a tert-butyl or benzyl thioether) or thioesters (for example a SC(xe2x95x90O)C1-C6 alkyl thioester).
There are several actual or potential chiral centres in the compounds according to the invention because of the presence of asymmetric carbon atoms. The presence of several asymmetric carbon atoms gives rise to a number of diastereoisomers with R or S stereochemistry at each chiral centre. The invention includes all such diastereoisomers and mixtures thereof. Currently, the preferred stereoconfiguration of the carbon atom carrying the R2 group is R; that of the carbon atom carrying the R4 group (when asymmetric) is S; and that of the carbon atom carrying the R1 group (when asymmetric) is R.
In the compounds of formula (I) as defined above for use according to the invention, and in the novel compounds of the invention of formula (II) as defined above (but subject to the provisos therein):
R1 may be, for example, hydrogen, methyl, or trifluoromethyl. Hydrogen is currently preferred.
R2 may be, for example:
optionally substituted C1-C8 alkyl, C3-C6 alkenyl, C3-C6 alkynyl or cycloalkyl;
phenyl(C1-C6 alkyl)-, phenyl(C3-C6 alkenyl)- or phenyl(C3-C6 alkynyl)-optionally substituted in the phenyl ring;
cycloalkyl(C1-C6 alkyl)-, cycloalkyl(C3-C6 alkenyl)- or cycloalkyl(C3-C6 alkynyl)-optionally substituted in the cycloalkyl ring;
heterocyclyl(C1-C6 alkyl)-, heterocyclyl(C3-C6 alkenyl)- or heterocyclyl(C3-C6 alkynyl)- optionally substituted in the heterocyclyl ring; or CH3(CH2)pO(CH2)qxe2x80x94 or CH3(CH2)pS(CH2)qxe2x80x94, wherein p is 0, 1, 2 or 3 and q is 1, 2 or 3.
Specific examples of R2 groups include
methyl, ethyl, n- and iso-propyl, n- and iso-butyl, n-pentyl, iso-pentyl 3-methyl-but-1-yl, n-hexyl, n-heptyl, n-acetyl, n-octyl, methylsulfanylethyl, ethylsulfanylmethyl, 2-methoxyethyl, 2-ethoxyethyl, 2-ethoxymethyl, 3-hydroxypropyl, allyl, 3-phenylprop-3-en-1-yl, prop-2-yn-1-yl, 3-phenylprop-2-yn-1-yl, 3-(2-chlorophenyl)prop-2-yn-1-yl, but-2-yn-1-yl, cyclopentyl, cyclohexyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, furan-2-ylmethyl, furan-3-methyl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-2-ylmethyl, piperidinylmethyl, phenylpropyl, 4-chlorophenylpropyl, 4-methylphenylpropyl, 4-methoxyphenylpropyl, benzyl, 4-chlorobenzyl, 4-methylbenzyl, and 4-methoxybenzyl.
Presently preferred groups at R2 are n-propyl, n-butyl, n-pentyl, benzyl and cyclopentylmethyl.
In the case of R3, hydrogen is presently preferred.
R4 may be, for example
the characterising group of a natural xcex1 amino acid, for example benzyl, or 4-methoxyphenylmethyl, in which any functional group may be protected, any amino group may be acylated and any carboxyl group present may be amidated; or  a group xe2x80x94[Alk]nR9 where Alk is a (C1-C6)alkylene or (C2-C6)alkenylene group optionally interrupted by one or more xe2x80x94Oxe2x80x94, or xe2x80x94Sxe2x80x94 atoms or xe2x80x94N(R12)xe2x80x94 groups [where R12 is a hydrogen atom or a (C1-C6)alkyl group], n is 0 or 1, and R9 is hydrogen or an optionally substituted phenyl, aryl, heterocyclyl, cycloalkyl or cycloalkenyl group or (only when n is 1) R9 may additionally be hydroxy, mercapto, (C1-C6)alkylthio, amino, halo, trifluoromethyl, nitro, xe2x80x94COOH, xe2x80x94CONH2, xe2x80x94COORA, xe2x80x94NHCORA, xe2x80x94CONHRA, NHRA, NRARB, or CONRARBwherein RA and RB are independently a (C1-C6)alkyl group; or
a benzyl group substituted in the phenyl ring by a group of formula xe2x80x94OCH2COR8 where R8 is hydroxyl, amino, (C1-C6)alkoxy, phenyl(C1-C6)alkoxy, (C1-C6)alkylamino, di((C1-C6)alkyl)amino, phenyl(C1-C6)alkylamino; or
a heterocyclic(C1-C6)alkyl group, either being unsubstituted or mono- or di-substituted in the heterocyclic ring with halo, nitro, carboxy, (C1-C6)alkoxy, cyano, (C1-C6)alkanoyl, trifluoromethyl (C1-C6)alkyl, hydroxy, formyl, amino, (C1-C6)alkylamino, di-(C1-C6)alkylamino, mercapto, (C1-C6)alkylthio, hydroxy(C1-C6)alkyl, mercapto(C1-C6)alkyl or (C1-C6)alkylphenylmethyl; or
a group xe2x80x94CRaRbRc in which:
each of Ra, Rb and Rc is independently hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(C1-C6)alkyl, (C3-C8)cycloalkyl; or
Rc is hydrogen and Ra and Rb are independently phenyl or heteroaryl such as pyridyl; or
Rc is hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(C1-C6)alkyl, or (C3-C8)cycloalkyl, and Ra and Rb together with the carbon atom to which they are attached form a 3 to 8 membered cycloalkyl or a 5- to 6-membered heterocyclic ring; or
Ra, Rb and Rc together with the carbon atom to which they are attached form a tricyclic ring (for example adamantyl); or
Ra and Rb are each independently (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(C1-C6)alkyl, or a group as defined for Rc below other than hydrogen, or Ra and Rb together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic ring, and Rc is hydrogen, xe2x80x94OH, xe2x80x94SH, halogen, xe2x80x94CN, xe2x80x94CO2H, (C1-C4)perfluoroalkyl, xe2x80x94CH2OH, xe2x80x94CO2(C1-C6)alkyl, xe2x80x94O(C1-C6)alkyl, xe2x80x94O(C2-C6)alkenyl, xe2x80x94S(C1-C6)alkyl, xe2x80x94SO(C1-C6)alkyl, xe2x80x94SO2(C1-C6) alkyl, xe2x80x94S(C2-C6)alkenyl, xe2x80x94SO(C2-C6)alkenyl, xe2x80x94SO2(C2-C6)alkenyl or a group xe2x80x94Qxe2x80x94W wherein Q represents a bond or xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94 or xe2x80x94SO2xe2x80x94 and W represents a phenyl, phenylalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkylalkyl, (C4-C8)cycloalkenyl, (C4-C8)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, which group W may optionally be substituted by one or more substituents independently selected from, hydroxyl, halogen, xe2x80x94CN, xe2x80x94CO2H, xe2x80x94CO2(C1-C6)alkyl, xe2x80x94CONH2, xe2x80x94CONH(C1-C6)alkyl, xe2x80x94CONH(C1-C6alkyl)2, xe2x80x94CHO, xe2x80x94CH2OH, (C1-C4)perfluoroalkyl, xe2x80x94O(C1-C6)alkyl, xe2x80x94S(C1-C6)alkyl, xe2x80x94SO(C1-C6)alkyl, xe2x80x94SO2(C1-C6)alkyl, -NO2, xe2x80x94NH2, xe2x80x94NH(C1-C6)alkyl, xe2x80x94N((C1-C6)alkyl)2, xe2x80x94NHCO(C1-C6)alkyl, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C3-C8)cycloalkyl, (C4-C8)cycloalkenyl, phenyl or benzyl.
Examples of particular R4 groups include methyl, ethyl, benzyl, 4-chlorobenzyl, 4-hydroxybenzyl, phenyl, cyclohexyl, cyclohexylmethyl, pyridin-3-ylmethyl, tert-butoxymethyl, naphthylmethyl, iso-butyl, sec-butyl, tert-butyl, 1-benzylthio-1-methylethyl, 1-methylthio-1-methylethyl, 1-mercapto-1-methylethyl, 1-methoxy-1-methylethyl, 1-hydroxy-1-methylethyl, 1-fluoro-1-methylethyl, hydroxymethyl, 2-hydroxethyl, 2-carboxyethyl, 2-methylcarbamoylethyl, 2-carbamoylethyl, and 4-aminobutyl. Presently preferred R4 groups include tert-butyl, iso-butyl, benzyl and methyl.
R3 and R4 when taken together with the nitrogen and carbon atoms to which they are respectively attached may form an optionally substituted saturated heterocyclic ring of 5 to 8 atoms. For example, R3 and R4 may form a bridge between the nitrogen and carbon atoms to which they are attached, said bridge being represented by the divalent radical xe2x80x94(CH2)3-6xe2x80x94, or xe2x80x94(CH2)rxe2x80x94Oxe2x80x94(CH2)sxe2x80x94, or xe2x80x94(CH2)rxe2x80x94Sxe2x80x94(CH2)sxe2x80x94, wherein r and s are each independently 1, 2 or 3 with the proviso that r+s =2, 3, 4, or 5.
R5 and R6 may independently be, for example, hydrogen, methyl, ethyl, tert-butyl, cyclopentyl, cyclohexyl, 1,1,3,3-tetramethylbutyl,benzyl, or 2-hydroxyethyl; or R5 and R6 when taken together with the nitrogen atom to which they are attached may form a saturated 5- to 8-membered monocyclic N-heterocyclic ring which is attached via the N atom and which optionally contains xe2x80x94N(R11)xe2x80x94 wherein R11 is hydrogen or C1-C6 alkyl, benzyl, acyl, or an amino protecting group, O, S, SO or SO2 as a ring member, and/or is optionally substituted on one or more C atoms by hydroxy, C1-C6 alkyl, hydroxy(C1-C6 alkyl)-, C1-C6 alkoxy, oxo, ketalised oxo, amino, mono(C1-C6 alkyl)amino, di(C1-C6 alkyl)amino, carboxy, C1-C6 alkoxycarbonyl, hydroxymethyl, C1-C6 alkoxymethyl, carbamoyl, mono(C1-C6 alkyl)carbamoyl, di(C1-C6 alkyl)carbamoyl, or hydroxyimino.
Examples of such rings are substituted or unsubstituted 1-pyrrolidinyl, piperidin-1-yl, 1-piperazinyl, hexahydro-1-pyridazinyl, morpholin-4-yl, tetrahydro-1,4-thiazin-4-yl, tetrahydro-1,4-thiazin-4-yl 1-oxide, tetrahydro-1,4-thiazin-4-yl 1,1-dioxide, hexahydroazipino, or octahydroazocino. Substituted examples of the foregoing are 2-(methylcarbamoyl)-1-pyrrolidinyl, 2-(hydroxymethyl)-1-pyrrolidinyl, 4-hydroxypiperidino, 2-(methylcarbamoyl)piperidino, 4-hydroxyiminopiperidino, 4-methoxypiperidino, 4-methylpiperidin-1yl, 4-benzylpiperidin-1-yl, 4-acetylpiperidin-1-yl,4-methyl-1-piperazinyl, 4-phenyl-1-piperazinyl, 1,4-dioxa-8-azaspiro[4,5]decan-8-yl, hexahydro-3-(methylcarbamoyl)-2-pyridazinyl, and hexahydro-1-(benzyloxycarbonyl)-2-pyridazinyl, decahydroisoquinolin-2-yl, and 1,2,3,4-tetrahydroisoquinolin-2-yl.
When A is a group of formula (IA), it is currently preferred that R5 be methyl or hydrogen, and R6 be methyl.
R7 may be, for example, hydrogen, or a group R20C(O)xe2x80x94 where R20 is a (C1-C6)alkyl group such as methyl or ethyl.
Specific examples of compounds useful as antibacterial agents in accordance with the invention include those of the specific Examples herein. Preferred novel compounds of the invention include
2R (or S)-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid (1S-dimethylcarbamoyl-ethyl)-amide and
2R (or S)-[(Formyl-hydroxy-amino)-methyl]-3-cyclopentyl-propionic acid (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl)-amide
and their pharmaceutically and veterinarily acceptable salts.
Compounds with which the invention is concerned the invention may be prepared by deprotecting an O-protected N-formyl-N-hydroxyamino compound of formula (II): 
in which R1, R2, and A are as defined in general formula (I) and R25 is a hydroxy protecting group removable to leave a hydroxy group by hydrogenolysis or hydrolysis. Benzyl is a preferred R25 group for removal by hydrogenolysis, and tert-butyl and tetrahydropyranyl are preferred groups for removal by acid hydrolysis.
Compounds of formula (II) wherein A is a group of formula (IA), (IB), (IC) or (ID) may be prepared by causing an acid of formula (III) or an activated derivative thereof to react with an amine of formula (IVA), (IVB), (IVC) or (IVD) respectively 
wherein R1 R2, R3, R4, R5, R6 and R7 are as defined in general formula (I) except that the xe2x80x94OH group in (IVB) and any substituents in R1 R2, R3, R4, R5, R6 and R7 which are potentially reactive in the coupling reaction may themselves be protected from such reaction, and R25 is as defined in relation to formula (II) above, and optionally removing protecting groups from the xe2x80x94OH group in (IVB) and R1 R2, R3, R4, R5, R6 and R7.
Compounds of formula (III) may be prepared by N-formylation, for example using acetic anhydride and formic acid, or 1-formylbenzotriazole, of compounds of formula (V) 
wherein R1, R2 and R25 are as defined in relation to formula (II) and X is either a chiral auxiliary or an OR26 group wherein R26 is hydrogen or a hydroxy protecting group. In the case where X is an OR26 group or a chiral auxiliary the hydroxy protecting group or auxiliary is removed after the formylation step to provide the compound of formula (V). Suitable chiral auxiliaries include substituted oxazolidinones which may be removed by hydrolysis in the presence of base.
In an alternative procedure compounds of general formula (II) may be prepared by N-formylation, for example using acetic anhydride and formic acid, or 1-formylbenzotriazole, of compounds of formula (VI) 
wherein R1, R2, R25 and A are as defined in relation to formula (II).
Compounds of formula (VI) wherein A is a group of formula (IA), (IB), (IC) or (ID) may be prepared by causing an acid of general formula (VII) or an activated derivative thereof 
wherein R1, R2 and R25 are as defined in relation to formula (II) to react with an amine of formula (IVA), (IVB), (IVC) or (IVD) respectively as defined above.
Alternatively compounds of general formula (VI) may be prepared by reduction of an oxime of general formula (VIII). 
Reducing agents include certain metal hydrides (e.g. sodium cyanoborohydride in acetic acid, triethylsilane or borane/pyridine) and hydrogen in the presence of a suitable catalyst.
In an alternative procedure compounds of general formula (II) wherein R1 and R2 are as defined in general formula (I), R25 is a hydroxy protecting group as defined above and A is a group of formula (IA) wherein R3, R4, R5 are as defined in general formula (IA) and R6 is hydrogen may be prepared by a 4-component Ugi reaction of carboxylic acid of general formula (III) as defined above, an amine of formula (IX), an aldehyde of formula (X) and an isonitrile of formula (XI)
R3xe2x80x94NH2xe2x80x83xe2x80x83(IX)
R4xe2x80x94CHOxe2x80x83xe2x80x83(X)
R5xe2x80x94CNxe2x80x83xe2x80x83(XI)
wherein R3, R4 and R5 are as defined above.
A compound of general formula (V) may be prepared by reduction of an oxime of general formula (XI) 
wherein R1, R2, and R25 are as defined above, and X is either an OR26 group as defined above or a chiral auxiliary. Reducing agents include certain metal hydrides (eg sodium cyanoborohydride in acetic acid, triethylsilane or borane/pyridine) and hydrogen in the presence of a suitable catalyst. Following the reduction when the group X is a chiral auxiliary it may be optionally converted to a OR26 group.
A compound of general formula (XI) can be prepared by reaction of a xcex2-keto carbonyl compound of general formula (XII) 
wherein R1, R2, and X are as defined above, with an O-protected hydroxylamine.
xcex2-keto carbonyl compounds (XII) may be prepared in racemic form by formylation or acylation of a carbonyl compound of general formula (XIII) 
wherein R2 and X are as defined above, with a compound of general formula (XIV) 
wherein R1 is as defined above and Z is a leaving group such as halogen or alkoxy, in the presence of a base.
Another method for the preparation of a compound of general formula (V) is by Michael addition of a hydroxylamine derivative to xcex1,xcex2-unsaturated carbonyl compounds of general formula (XV) 
wherein R1, R2, and X are as defined above. Following the Michael addition reaction, when the group X is a chiral auxiliary it may be optionally converted to a OR26 group. The xcex1,xcex2-unsaturated carbonyl compounds (XV) may be prepared by standard methods.
Salts of the compounds of the invention include physiologically acceptable acid addition salts for example hydrochlorides, hydrobromides, sulphates, methane sulphonates, p-toluenesulphonates, phosphates, acetates, citrates, succinates, lactates, tartrates, fumarates and maleates. Salts may also be formed with bases, for example sodium, potassium, magnesium, and calcium salts.
Compositions with which the invention is concerned may be prepared for administration by any route consistent with the pharmacokinetic properties of the active ingredient(s).
Orally administrable compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
For topical application to the skin, the active ingredient(s) may be made up into a cream, lotion or ointment. Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.
The active ingredient(s) may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle. Intra-venous infusion is another route of administration for the compounds used in accordance with the invention.
Safe and effective dosages for different classes of patient and for different disease states will be determined by clinical trial as is required in the art. It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
The finding that compounds with PDF inhibitory activity can inhibit or prevent bacterial growth, opens up a novel approach for identifying new antibacterial agents by screening test compounds for activity as inhibitors of PDF in vitro, followed by confirmation of their antibacterial ability using bacterial growth inhibition studies. This finding also makes available (i) the use of compounds with PDF inhibitory activity as antibacterial agents, and (ii) a method for the treatment of bacterial infection or contamination by applying or administering a compound which inhibits the activity of bacterial PDF.
According to a further aspect of the invention therefore, there is provided a method for the identification of antibacterial compounds, comprising screening test compounds for their ability to inhibit PDF in vitro, selecting those compounds which exhibit said ability and testing these for their ability to inhibit bacterial growth. The ability to inhibit bacterial growth can be performed using classical plate or broth culture bacterial growth inhibition studies, such as those performed in the Biological Examples herein.
A suitable in vitro PDF inhibition screen may comprise mixing together PDF, a PDF substrate, preferably, labelled with a detectable marker, and the test compound and assessing after a suitable length of time whether or not the presence of the test compound inhibits the ability of PDF to deformylate the substrate.
In a preferred embodiment, the cleaved substrate is detected with a fluorogenic marker such as fluorescamine. On removal of the formyl group from the N-terminal methionine of the PDF substrate, the free amino group reacts with fluorescamine generating a fluorescent product.
An alternative screen involves assessing whether a protein expressed by bacteria that express endogenous (or recombinantly expressed) PDF, when grown in the presence of a test compound, yields suitable substrate for N-terminal sequencing, or yields a lesser amount of substrate, than protein expressed from the same bacteria grown in the absence of the test compound. Such a method could be based on that used in the Biological Examples herein.
The person skilled in the art will be able to develop, without inventive input, alternative methods for screening test compounds for their ability to inhibit bacterial PDF.
The natural antibiotic actinonin (see for example J. C. S Perkin I, 1975, 819) is a hydroxamic acid derivative of Structure (A): 
In addition to actinonin, various structural analogues of actinonin have also been shown to have antibacterial activity (see for example Broughton et al. (Devlin et al. Journal of the Chemical Society. Perkin Transactions 1 (9):830-841, 1975; Broughton et al. Journal of the Chemical Society. Perkin Transactions 1 (9):857-860, 1975).
To date, however, the mechanism underlying the antibacterial activity of actinonin has not been known. The present inventors have found that actinonin inhibits the activity of bacterial PDF.
The matlystatin group of compounds share a number of structural similarities with actinonin. Both are peptidic molecules with functional hydroxamic acid metal binding groups (Ogita et al., J. Antibiotics. 45(11):1723-1732; Tanzawa et al., J. Antibiotics. 45(11):1733-1737; Haruyama et al., J. Antibiotics. 47(12):1473-1480; Tamaki et al., J. Antibiotics. 47(12):1481-1492). The matlystatins and their close structural analogues are characterised by the presence in the molecule of a divalent piperazin-1,6-diyl group, i.e. 
In view of their close structural similarity to actinonin, the observation that actinonin inhibits PDF implies that matlystatin compounds may also inhibit PDF.
According to a further aspect of the present invention there is provided the use of a compound which inhibits the activity of bacterial PDF, in the preparation of an antibacterial composition or agent, provided that (i) the compound is not of formula (XI)
RCOxe2x80x94CH(W)xe2x80x94NHxe2x80x94COxe2x80x94CH(Y)xe2x80x94CH2xe2x80x94COxe2x80x94NHxe2x80x94OHxe2x80x83xe2x80x83(XI)
wherein,
(a) R is a cyclic amino group, W is hydrogen, methyl, isopropyl, isobutyl or benzyl, and Y is hydrogen, C1-C6 alkyl, phenyl, benzyl, 4-chlorophenylmethyl, 4-nitrophenylmethyl, or 4-aminophenylmethyl; or,
(b) R is 2-pyridylamino or 2-thiazolylamino, W is isopropyl and Y is n-pentyl; or,
(c) R is diethylamino, W is methyl or isopropyl and Y is n-pentyl;
or (ii) the compound is not one containing a divalent piperazin-1,6-diyl group, i.e. a group of formula (XII): 
According to a further aspect of the invention there is provided a method of treating bacterial infection or contamination by administering to a patient suffering such infection or contamination, or applying to the site of such infection or contamination, an antibacterially effective amount of a compound which inhibits the activity of bacterial PDF enzyme, provided that the compound is not one provided in the provisos in the immediately preceeding paragraph.
These provisos exclude actinonin and its antibacterially active analogues as disclosed in Devlin et al., Journal of the Chemical Society. Perkin Transactions 1 (9):830-841,1975 and Broughton et al. Journal of the Chemical Society. Perkin Transactions 1 (9):857-860, 1975, and the matlystatin compounds disclosed in Ogita et al., J. Antibiotics. 45(11):1723-1732; Tanzawa et al., J. Antibiotics. 45(11):1733-1737; Haruyama et al., J. Antibiotics. 47(12):1473-1480 and Tamaki et al., J. Antibiotics. 47(12):1481-1492.
The following examples illustrate embodiments of the invention. L-tert-Leucine-N-methylamide and L-tert-leucine-N,N-dimethylamide and other amino acid derivatives were prepared according to established literature methods.
The following abbreviations have been used throughout:
DMF N,N-Dimethylformamide
EDC N-Ethyl-Nxe2x80x2-(3-dimethylaminopropyl)carbodiimide hydrochloride
HOAt 1-Hydroxy-7-aza-benzotriazole
HOBt 1-Hydroxybenzotriazole
HPLC High performance liquid chromatography
LRMS Low resolution mass spectrometry
TLC Thin layer chromatography
1H and 13C NMR spectra were recorded using a Bruker AC250E spectrometer at 250.1 and 62.9 MHz, respectively. Mass spectra were obtained using a Perkin Elmer Sciex API 165 spectrometer using both positive and negative ion modes. Infra-red spectra were recorded on a Perkin Elmer PE 1600 FTIR spectrometer.